Method of selecting antioxidants for use in topically applied compositions
View Patent ↗Antioxidant-containing compositions and methods for confirming antioxidant activity of a composition formulated for topical application to skin. Methods for testing a composition for ability to inhibit both ultraviolet radiation-induced lipid peroxidation on skin and ultraviolet radiation-induced reactive oxygen species formation in the stratum corneum. Compositions and methods for treating and preventing photodamage to skin.
1. An ex vivo method for screening compounds for antioxidant behavior in a composition to be topically applied to skin, the method comprises determining the compound's ability both to inhibit ultraviolet radiation-induced skin lipid hydroperoxide formation and to inhibit ultraviolet radiation-induced reactive oxygen species formation in the epidermis;
wherein determining inhibition of ultraviolet radiation-induced reactive oxygen species formation in the epidermis comprises applying the composition containing said compound(s) to a surface of a skin tissue; treating the skin tissue with dihydrorhodamine, irradiating the treated skin tissue with a UV source; and imaging the skin tissue using two-photon fluorescence intensity imaging; wherein a base formula without said compound was used as a control;
and wherein determining inhibition of UVR-induced skin lipid hydroperoxide formation comprises the steps of applying to distinct areas of skin of a subject an antioxidant-containing composition and a placebo composition to produce an antioxidant skin site containing antioxidant and skin lipids and a placebo skin site containing placebo and skin lipids; applying a strip to the antioxidant skin site and the placebo skin site to produce an antioxidant strip sample containing antioxidant and skin lipids and a placebo strip sample containing placebo and skin lipids; removing said strip samples from the skin and exposing said strip samples to UVR to form a UVR-induced antioxidant/lipid reaction product on the antioxidant strip sample and a UVR-induced placebo/lipid reaction product on the placebo strip sample; separately contacting the antioxidant strip sample and the placebo strip sample with solvent to prepare a first extract containing UVR-induced antioxidant/lipid reaction product and a second extract containing UVR-induced placebo/lipid reaction product; assaying said first and second extracts for lipid hydroperoxide content for each extract; and comparing the lipid hydroperoxide content of the first extract to the lipid hydroperoxide content of the second extract.
2. The method of claim 1 , wherein determining inhibition of UVR-induced skin lipid hydroperoxide formation comprises the further steps of applying placebo to two distinct sites on the skin of the subject; producing strip samples from each site; subjecting strip samples from only one of the two placebo sites to UVR to produce a subset of irradiated placebo strip samples and a subset of nonirradiated placebo strip samples; separately contacting nonirradiated placebo strip samples with solvent to prepare a third extract containing placebo and skin lipids; and assaying said third extract for lipid hydroperoxide content to determine background lipid hydroperoxide formation.
3. The method of claim 2 , wherein comparing the lipid hydroperoxide content of the first extract to the lipid hydroperoxide content of the second extract comprises calculating percent lipid hydroperoxide formation by the following formula:
% LF =((LOOH1−LOOH3)/(LOOH2−LOOH3))×100
wherein % LF is the percent lipid hydroperoxide formation, LOOH1 is the lipid hydroperoxide content of the first extract, LOOH2 is the lipid hydroperoxide content of the second extract, and LOOH3 is the lipid hydroperoxide content of the third extract.